Psychostimulants increase transmission of the neurotransmitter, dopamine, in the nucleus accumbens and prefrontal cortex. This action contributes to the rewarding effects of these agents and the initiation of drug abuse. Following continued drug use, enduring changes in brain chemistry are observed within the nucleus accumbens, prefrontal cortex and other regions of the prefrontal-cortico-striatal loop, a circuit that controls incentive motivation, learning and impulsivity. Increasing evidence suggests that these neuroadaptations lead to the dysregulation of behavior that characterizes addiction. Psychostimulants enhance dopamine transmission by inhibiting the dopamine transporter, a protein that clears dopamine released into the synaptic cleft. By inhibiting dopamine clearance, synaptic and extracellular neurotransmitter concentrations are increased. We previously provided evidence that synthetic kappa opioid receptor agonists inhibit dopamine transmission within the nucleus accumbens and striatum by decreasing release and facilitating dopamine transporter function. Their coadministration with cocaine attenuates the behavioral and neurochemical effects of this stimulant. We have hypothesized that the cocaine-antagonist like effect of kappa opioid agonists may result from their ability to regulate the dopamine transporter. At present, however, the cellular mechanism(s) mediating the interaction of kappa opioid receptors with the dopamine transporter is unknown. We have used heterologous expression systems and synaptosomal preparations to begin to address this issue. Live cell imaging of cells co-expressing the kappa opioid receptor and the dopamine transporter revealed that activation of kappa opioid receptors by synthetic agonists produces a rapid and pertussis sensitive up-regulation of transporter function. Similar effects are observed in response to salvinorin A, a naturally occurring, high potency kappa opioid receptor agonist. By examining the effects of agonists in the presence of various kinase inhibitors, we have identified a critical role of a specific kinase in mediating the kappa opioid receptor upregulation of transporter function. Consistent with the presence of consensus sites for this kinase in a restricted location of the transporter, we have found that truncation or single point mutation of the dopamine transporter, so as to remove these sites, results in a loss of kappa opioid receptor regulation of transport. The finding that inhibitors of this kinase microinjected into the nucleus accumbens attenuate the behavioral effects of kappa opioid receptor agonists suggest that activation of this kinase contributes to the pharmacological actions of these agents. On-going studies seek to elucidate the cellular mechanisms by which KOPr systems regulate monoamine transporters and whether blockade of this regulation attenuates the cocaine-antagonist like actions of KOPr agonists. In studies conducted at the systems level, we have begun to define the role of kappa opioid receptors in regulating dopamine transmission in the prefrontal-cortico-striatal loop. Using microdialysis we have demonstrated that kappa opioid receptor located in the prefrontal cortex tonically inhibit the basal activity of dopamine neurons projecting to this region. Kappa opioid receptors are also located in the ventral tegmental area where they selectively regulate the activity of dopamine neurons projecting to the prefrontal cortex. In contrast, however, to kappa opioid receptors located at the level of the dopamine nerve terminal, kappa opioid receptors located at the level of the cell body are not tonically active. Mu opioid receptors (MOR) are enriched in the ventral tegmental area(VTA),the site of origin of the mesocorticolimbic dopamine system. This dopamine system is implicated in mediating the reinforcing effects of natural rewards and drugs of abuse. The VTA is a critical site mediating the rewarding effects of MOR agonists. MOR activation in this region increases dopamine transmission in the nucleus accumbens. This action is thought to contribute to the rewarding effect of MOR agonists. Morphological data suggest that MOR are located on non-dopaminergic neurons in the VTA. Intracellular recordings in slice preparations of the VTA revealed that morphine increases the firing rate of dopamine neurons but inhibits the firing rate of non-dopaminergic neurons. Although the identity of the non-dopaminergic neurons was not definitively determined, these findings led to the hypothesis that activation of MOR on GABA neurons inhibits their activity, thereby, decreasing GABA release and disinhibiting VTA DA neurons. As a consequence DA release in the NAc is increased. However, the data upon which this hypothesis is based were obtained in a slice preparation in which connectivity of functional circuits is not preserved. Therefore, question exist as to whether MOR activation affects GABA release in awake animals. Alterations in glutamate transmission are recognized to play an important role in shaping the pattern of DA neuronal activity in the brain. Only two studies have examined opioid regulation of glutamate transmission in the VTA. In these, slice preparations from halothane or isoflurane anesthetized animals were used. Importantly, however, general anesthetics may affect impulse activity, as well as basal glutamate (and GABA) transmission. To date, studies examining MOR regulation of VTA glutamate transmission in the awake animal are lacking. Furthermore, no studies have simultaneously evaluated the influence of MOR agonists on GABA, glutamate and dopamine transmission in the VTA Using microdialysis in the awake and behaving animal, we have demonstrated that reverse dialysis of a highly selective MOR agonist into the VTA produces a concentration-dependent increase in dialysate DA concentrations. Basal dopamine overflow in the VTA was unaltered in mice lacking the MOR gene confirming this effect results from the specific activation of MOR in this region. In contrast to dopamine, basal GABA overflow in mice lacking the MOR gene was significantly increased, while glutamate overflow was decreased. Intra-VTA perfusion of DAMGO to wildtype mice increased dopamine overflow. GABA concentrations were decreased whereas glutamate concentrations in the VTA were unaltered. These data provide the first direct demonstration of tonically active MOR systems in the VTA that regulate basal glutamatergic and GABAergic neurotransmission in this region. We hypothesize that increased GABAergic neurotransmission following constitutive deletion of MOR is due to the elimination of a tonic inhibitory influence of MOR on GABA neurons in the VTA, whereas decreased glutamatergic neurotransmission in mice lacking the MOR gene is a consequence of intensified GABA tone on glutamatergic neurons and/or terminals. As a consequence, somatodendritic dopamine release is unaltered. Together our findings indicate a critical role of VTA MOR in maintaining an intricate balance between excitatory and inhibitory inputs to dopaminergic neurons. Furthermore, they provide suggestive evidence that VTA MOR may modulate vulnerability to drugs of abuse by regulating GABA and glutamatergic inputs to dopaminergic neurons. Studies testing this hypothesis are currently in progress.